Abstract Male fertility is dependent on the successful production of both reproductive hormones and mature sperm, which requires the concerted activity of germ cells, supporting somatic cells, and the hypothalamic-pituitary axis. The diversity of cell types involved in these processes in the testis has largely been defined histologically, but our molecular understanding of somatic populations is largely limited by the availability of transgenic lines. Therefore, to achieve an unbiased analysis of the somatic compartment and molecular cell types, I have applied single cell RNA-seq to the adult mouse testis. Transcriptome analysis of ~5000 single-cells identified all major somatic cell types, and a previously unappreciated interstitial cell population (Tcf21+ cells). Clustering and principle component analyses suggest that the novel cell population is related to both myoid and Leydig cells. Marker gene analysis of this population is consistent with this population being a mesenchymal stem cell (MSC). In other organs, MSCs are known for their regenerative and immunomodulatory potential, but whether this cell population is capable of supporting testicular tissue homeostasis and regeneration in the adult testis, or serving as the cell of origin myiod or Leydig cells, remains unknown. Therefore, this proposal aims to explore the functional role of Tcf21+ cells in the embryonic and adult testis. Based on my preliminary data and previous genetic studies demonstrating a key role for Tcf21 in testis differentiation and development, I hypothesize that Tcf21+ cells are precursors for myoid and Leydig cells, and serve as a reserve somatic stem cell in the adult testis. To address these questions, I will use a combination of genetic lineage tracing, immunohistochemical and functional characterization, and in vivo targeted cell ablation. These studies will elucidate the development and homeostatic function of interstitial cells in the testis, and will provide new evidence for a reserve somatic stem cell population, which could be used to inform new therapeutic approaches for patients with hypoandrogenism and/or infertility. Additionally, completion of this work will provide me with critical scientific, technical, and medical training, as outlined in my training plan, which will be foundational for a successful career as a physician scientist studying the intrinsic and extrinsic regulation of spermatogenesis and male fertility.